Overexpression of ANAC046 Promotes Suberin Biosynthesis in Roots of Arabidopsis thaliana.

NAC (NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped cotyledon)) proteins are one of the largest families of plant-specific transcription factors, and this family is present in a wide range of land plants. Here, we have investigated the role of ANAC046 in the regulation of suberin biosynthesis and deposition in Arabidopsis. Subcellular localization and transcriptional activity assays showed that ANAC046 localizes in the nucleus, where it functions as a transcription activator. Analysis of the PANAC046:GUS lines revealed that ANAC046 is mainly expressed in the root endodermis and periderm, and is also induced in leaves by wounding. The transgenic lines overexpressing ANAC046 exhibited defective surfaces on the aerial plant parts compared to the wild-type (WT) as characterized by increased permeability for Toluidine blue stain and greater chlorophyll leaching. Quantitative RT-PCR analysis showed that the expression of suberin biosynthesis genes was significantly higher in the roots and leaves of overexpression lines compared to the WT. The biochemical analysis of leaf cuticular waxes showed that the overexpression lines accumulated 30% more waxes than the WT. Concurrently, overexpression lines also deposited almost twice the amount of suberin content in their roots compared with the WT. Taken together, these results showed that ANAC046 is an important transcription factor that promotes suberin biosynthesis in Arabidopsis thaliana roots.

Overexpression of miR169o, an Overlapping MicroRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice.

Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs (miRNAs) in this phenomenon, 63 differentially expressed overlapping miRNAs in response to Xoo infection and N limitation stress in rice were identified through deep RNA sequencing and stem-loop quantitative real-time PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than the wild type (WT). Transcript level assays showed that under different N supply conditions, miR169o oppositely regulated NRT2, and this is reduced under normal N supply conditions but remarkably induced under N-limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with the WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to BB in rice. Consistently, transient expression of NF-YA genes in rice protoplasts promoted the transcripts of PR genes and NRT2 genes, while it reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the co ordinated regulatory mechanisms of cross-talk between Xoo infection and N deficiency responses in rice.

Altered Expression of OsNLA1 Modulates Pi Accumulation in Rice (Oryza sativa L.) Plants.

Current agricultural practices rely on heavy use of fertilizers for increased crop productivity. However, the problems associated with heavy fertilizer use, such as high cost and environmental pollution, require the development of crop species with increased nutrient use efficiency. In this study, by using transgenic approaches, we have revealed the critical role of OsNLA1 in phosphate (Pi) accumulation of rice plants. When grown under sufficient Pi and nitrate levels, OsNLA1 knockdown (Osnla1-1, Osnla1-2, and Osnla1-3) lines accumulated higher Pi content in their shoot tissues compared to wild-type, whereas, over-expression lines (OsNLA1-OE1, OsNLA1-OE2, and OsNLA1-OE3) accumulated the least levels of Pi. However, under high Pi levels, knockdown lines accumulated much higher Pi content compared to wild-type and exhibited Pi toxicity symptoms in the leaves. In contrast, the over-expression lines had 50-60% of the Pi content of wild-type and did not show such symptoms. When grown under limiting nitrate levels, OsNLA1 transgenic lines also displayed a similar pattern in Pi accumulation and Pi toxicity symptoms compared to wild-type suggesting an existence of cross-talk between nitrogen (N) and phosphorous (P), which is regulated by OsNLA1. The greater Pi accumulation in knockdown lines was a result of enhanced Pi uptake/permeability of roots compared to the wild-type. The cross-talk between N and P was found to be nitrate specific since the knockdown lines failed to over-accumulate Pi under low (sub-optimal) ammonium level. Moreover, OsNLA1 was also found to interact with OsPHO2, a known regulator of Pi homeostasis, in a Yeast Two-Hybrid (Y2H) assay. Taken together, these results show that OsNLA1 is involved in Pi homeostasis regulating Pi uptake and accumulation in rice plants and may provide an opportunity to enhance P use efficiency by manipulating nitrate supply in the soil.

Overexpression of OsGATA12 regulates chlorophyll content, delays plant senescence and improves rice yield under high density planting.

Agronomic traits controlling the formation, architecture and physiology of source and sink organs are main determinants of rice productivity. Semi-dwarf rice varieties with low tiller formation but high seed production per panicle and dark green and thick leaves with prolonged source activity are among the desirable traits to further increase the yield potential of rice. Here, we report the functional characterization of a zinc finger transcription factor, OsGATA12, whose overexpression causes increased leaf greenness, reduction of leaf and tiller number, and affects yield parameters. Reduced tillering allowed testing the transgenic plants under high density which resulted in significantly increased yield per area and higher harvest index compared to wild-type. We show that delayed senescence of transgenic plants and the corresponding longer stay-green phenotype is mainly due to increased chlorophyll and chloroplast number. Further, our work postulates that the increased greenness observed in the transgenic plants is due to more chlorophyll synthesis but most significantly to decreased chlorophyll degradation, which is supported by the reduced expression of genes involved in the chlorophyll degradation pathway. In particular we show evidence for the down-regulation of the STAY GREEN RICE gene and in vivo repression of its promoter by OsGATA12, which suggests a transcriptional repression function for a GATA transcription factor for prolonging the onset of senescence in cereals.

Genome-wide binding analysis of AtGNC and AtCGA1 demonstrates their cross-regulation and common and specific functions.

GATA transcription factors are involved in multiple processes in plant growth and development. Two GATA factors, NITRATE-INDUCIBLE,CARBON METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1, also named GNL), are important regulators in greening, flowering, senescence, and hormone signaling. However, their direct target genes related to these biological processes are poorly characterized. Here, GNC and CGA1 are shown to be transcription activators and by using chromatin immunoprecipitation sequencing (ChIP-seq), 1475 and 638 genes are identified to be associated with GNC and CGA1 binding, respectively. Enrichment of diverse motifs in the peak binding regions for GNC and CGA1 suggests the possibility that these two transcription factors also interact with other transcription factors and in addition genes coding for DNA-binding proteins are highly enriched among GNC- and CGA1-associated genes. Despite the fact that these two GATA factors are known to share a large portion of co-expressed genes, our analysis revealed a low percentage of overlapping binding-associated genes for these two homologues. This suggests a possible cross-regulation between these, which is verified using ChIP-qPCR. The common and specific biological processes regulated by GNC and CGA1 also support this notion. Functional analysis of the binding-associated genes revealed that those encoding transcription factors, E3 ligase, as well as genes with roles in plant development are highly enriched, indicating that GNC and CGA1 mediate complex genetic networks in regulating different aspects of plant growth and development.

NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

Expression of OsMYB55 in maize activates stress-responsive genes and enhances heat and drought tolerance.

BACKGROUND: Plant response mechanisms to heat and drought stresses have been considered in strategies for generating stress tolerant genotypes, but with limited success. Here, we analyzed the transcriptome and improved tolerance to heat stress and drought of maize plants over-expressing the OsMYB55 gene. RESULTS: Over-expression of OsMYB55 in maize decreased the negative effects of high temperature and drought resulting in improved plant growth and performance under these conditions. This was evidenced by the higher plant biomass and reduced leaf damage exhibited by the transgenic lines compared to wild type when plants were subjected to individual or combined stresses and during or after recovery from stress. A global transcriptomic analysis using RNA sequencing revealed that several genes induced by heat stress in wild type plants are constitutively up-regulated in OsMYB55 transgenic maize. In addition, a significant number of genes up-regulated in OsMYB55 transgenic maize under control or heat treatments have been associated with responses to abiotic stresses including high temperature, dehydration and oxidative stress. The latter is a common and major consequence of imposed heat and drought conditions, suggesting that this altered gene expression may be associated with the improved stress tolerance in these transgenic lines. Functional annotation and enrichment analysis of the transcriptome also pinpoint the relevance of specific biological processes for stress responses. CONCLUSIONS: Our results show that expression of OsMYB55 can improve tolerance to heat stress and drought in maize plants. Enhanced expression of stress-associated genes may be involved in OsMYB55-mediated stress tolerance. Possible implications for the improved tolerance to heat stress and drought of OsMYB55 transgenic maize are discussed.

Ammonium-induced architectural and anatomical changes with altered suberin and lignin levels significantly change water and solute permeabilities of rice (Oryza sativa L.) roots.

MAIN CONCLUSION: Non-optimal ammonium levels significantly alter root architecture, anatomy and root permeabilities for water and nutrient ions. Higher ammonium levels induced strong apoplastic barriers whereas it was opposite for lower levels. Application of nitrogen fertilizer increases crop productivity. However, non-optimal applications can have negative effects on plant growth and development. In this study, we investigated how different levels of ammonium (NH4 (+)) [low (30 or 100 µM) or optimum (300 µM) or high (1000 or 3000 µM)] affect physio-chemical properties of 1-month-old, hydroponically grown rice roots. Different NH4 (+) treatments markedly altered the root architecture and anatomy. Plants grown in low NH4 (+) had the longest roots with a weak deposition of suberised and lignified apoplastic barriers, and it was opposite for plants grown in high NH4 (+). The relative expression levels of selected suberin and lignin biosynthesis candidate genes, determined using qRT-PCR, were lowest in the roots from low NH4 (+), whereas, they were highest for those grown in high NH4 (+). This was reflected by the suberin and lignin contents, and was significantly lower in roots from low NH4 (+) resulting in greater hydraulic conductivity (Lp r) and solute permeability (P sr) than roots from optimum NH4 (+). In contrast, roots grown at high NH4 (+) had markedly greater suberin and lignin contents, which were reflected by strong barriers. These barriers significantly decreased the P sr of roots but failed to reduce the Lp r below those of roots grown in optimum NH4 (+), which can be explained in terms of the physical properties of the molecules used and the size of pores in the apoplast. It is concluded that, in rice, non-optimal NH4 (+) levels differentially affected root properties including Lp r and P sr to successfully adapt to the changing root environment.

Overexpression of the CC-type glutaredoxin, OsGRX6 affects hormone and nitrogen status in rice plants.

Glutaredoxins (GRXs) are small glutathione dependent oxidoreductases that belong to the Thioredoxin (TRX) superfamily and catalyze the reduction of disulfide bonds of their substrate proteins. Plant GRXs include three different groups based on the motif sequence, namely CPYC, CGFS, and CC-type proteins. The rice CC-type proteins, OsGRX6 was identified during the screening for genes whose expression changes depending on the level of available nitrate. Overexpression of OsGRX6 in rice displayed a semi-dwarf phenotype. The OsGRX6 overexpressors contain a higher nitrogen content than the wild type, indicating that OsGRX6 plays a role in homeostatic regulation of nitrogen use. Consistent with this, OsGRX6 overexpressors displayed delayed chlorophyll degradation and senescence compared to the wild type plants. To examine if the growth defect of these transgenic lines attribute to disturbed plant hormone actions, plant hormone levels were measured. The levels of two cytokinins (CKs), 2-isopentenyladenine and trans-zeatin, and gibberellin A1 (GA1) were increased in these lines. We also found that these transgenic lines were less sensitive to exogenously applied GA, suggesting that the increase in GA1 is a result of the feedback regulation. These data suggest that OsGRX6 affects hormone signaling and nitrogen status in rice plants.

OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis, transport and distribution.

Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier-like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed-setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) -localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin-related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.

Metabolic and co-expression network-based analyses associated with nitrate response in rice.

BACKGROUND: Understanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions. RESULTS: A gene co-expression network representing clusters of rice genes with similar expression patterns across four nitrogen conditions and two tissue types was generated. The resulting 18 clusters were analyzed for enrichment of significant gene ontology (GO) terms. Four clusters exhibited significant correlation with limiting and reducing nitrate treatments. Among the identified enriched GO terms, those related to nucleoside/nucleotide, purine and ATP binding, defense response, sugar/carbohydrate binding, protein kinase activities, cell-death and cell wall enzymatic activity are enriched. Although a subset of functional categories are more broadly associated with the response of rice organs to limiting N and N reduction, our analyses suggest that N reduction elicits a response distinguishable from that to adaptation to limiting N, particularly in leaves. This observation is further supported by metabolic profiling which shows that several compounds in leaves change proportionally to the nitrate level (i.e. higher in sufficient N vs. limiting N) and respond with even higher levels when the nitrate level is reduced. Notably, these compounds are directly involved in N assimilation, transport, and storage (glutamine, asparagine, glutamate and allantoin) and extend to most amino acids. Based on these data, we hypothesize that plants respond by rapidly mobilizing stored vacuolar nitrate when N deficit is perceived, and that the response likely involves phosphorylation signal cascades and transcriptional regulation. CONCLUSIONS: The co-expression network analysis and metabolic profiling performed in rice pinpoint the relevance of signal transduction components and regulation of N mobilization in response to limiting N conditions and deepen our understanding of N responses and N use in crops.

The challenges of commercializing second-generation transgenic crop traits necessitate the development of international public sector research infrastructure.

It has been 30 years since the first transformation of a gene into a plant species, and since that time a number of biotechnology products have been developed, with the most important being insect- and herbicide-resistant crops. The development of second-generation products, including nutrient use efficiency and tolerance to important environmental stressors such as drought, has, up to this time, been less successful. This is in part due to the inherent complexities of these traits and in part due to limitations in research infrastructure necessary for public sector researchers to test their best ideas. Here we discuss lessons from previous work in the generation of the first-generation traits, as well as work from our labs and others on identifying genes for nitrogen use efficiency. We then describe some of the issues that have impeded rapid progress in this area. Finally, we propose the type of public sector organization that we feel is necessary to make advances in important second-generation traits such as nitrogen use efficiency.

High throughput RNA sequencing of a hybrid maize and its parents shows different mechanisms responsive to nitrogen limitation.

BACKGROUND: Development of crop varieties with high nitrogen use efficiency (NUE) is crucial for minimizing N loss, reducing environmental pollution and decreasing input cost. Maize is one of the most important crops cultivated worldwide and its productivity is closely linked to the amount of fertilizer used. A survey of the transcriptomes of shoot and root tissues of a maize hybrid line and its two parental inbred lines grown under sufficient and limiting N conditions by mRNA-Seq has been conducted to have a better understanding of how different maize genotypes respond to N limitation. RESULTS: A different set of genes were found to be N-responsive in the three genotypes. Many biological processes important for N metabolism such as the cellular nitrogen compound metabolic process and the cellular amino acid metabolic process were enriched in the N-responsive gene list from the hybrid shoots but not from the parental lines' shoots. Coupled to this, sugar, carbohydrate, monosaccharide, glucose, and sorbitol transport pathways were all up-regulated in the hybrid, but not in the parents under N limitation. Expression patterns also differed between shoots and roots, such as the up-regulation of the cytokinin degradation pathway in the shoots of the hybrid and down-regulation of that pathway in the roots. The change of gene expression under N limitation in the hybrid resembled the parent with the higher NUE trait. The transcript abundances of alleles derived from each parent were estimated using polymorphic sites in mapped reads in the hybrid. While there were allele abundance differences, there was no correlation between these and the expression differences seen between the hybrid and the two parents. CONCLUSIONS: Gene expression in two parental inbreds and the corresponding hybrid line in response to N limitation was surveyed using the mRNA-Seq technology. The data showed that the three genotypes respond very differently to N-limiting conditions, and the hybrid clearly has a unique expression pattern compared to its parents. Our results expand our current understanding of N responses and will help move us forward towards effective strategies to improve NUE and enhance crop production.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

Nitrogen transporter and assimilation genes exhibit developmental stage-selective expression in maize (Zea mays L.) associated with distinct cis-acting promoter motifs.

Nitrogen is considered the most limiting nutrient for maize (Zea mays L.), but there is limited understanding of the regulation of nitrogen-related genes during maize development. An Affymetrix 82K maize array was used to analyze the expression of ≤ 46 unique nitrogen uptake and assimilation probes in 50 maize tissues from seedling emergence to 31 d after pollination. Four nitrogen-related expression clusters were identified in roots and shoots corresponding to, or overlapping, juvenile, adult, and reproductive phases of development. Quantitative real time PCR data was consistent with the existence of these distinct expression clusters. Promoters corresponding to each cluster were screened for over-represented cis-acting elements. The 8-bp distal motif of the Arabidopsis 43-bp nitrogen response element (NRE) was over-represented in nitrogen-related maize gene promoters. This conserved motif, referred to here as NRE43-d8, was previously shown to be critical for nitrate-activated transcription of nitrate reductase (NIA1) and nitrite reductase (NIR1) by the NIN-LIKE PROTEIN 6 (NLP6) in Arabidopsis. Here, NRE43-d8 was over-represented in the promoters of maize nitrate and ammonium transporter genes, specifically those that showed peak expression during early-stage vegetative development. This result predicts an expansion of the NRE-NLP6 regulon and suggests that it may have a developmental component in maize. We also report leaf expression of putative orthologs of nitrite transporters (NiTR1), a transporter not previously reported in maize. We conclude by discussing how each of the four transcriptional modules may be responsible for the different nitrogen uptake and assimilation requirements of leaves and roots at different stages of maize development.

Rice cytokinin GATA transcription Factor1 regulates chloroplast development and plant architecture.

Chloroplast biogenesis has been well documented in higher plants, yet the complex methods used to regulate chloroplast activity under fluctuating environmental conditions are not well understood. In rice (Oryza sativa), the CYTOKININ-RESPONSIVE GATA TRANSCRIPTION FACTOR1 (Cga1) shows increased expression following light, nitrogen, and cytokinin treatments, while darkness and gibberellin reduce expression. Strong overexpression of Cga1 produces dark green, semidwarf plants with reduced tillering, whereas RNA interference knockdown results in reduced chlorophyll and increased tillering. Coexpression, microarray, and real-time expression analyses demonstrate a correlation between Cga1 expression and the expression of important nucleus-encoded, chloroplast-localized genes. Constitutive Cga1 overexpression increases both chloroplast biogenesis and starch production but also results in delayed senescence and reduced grain filling. Growing the transgenic lines under different nitrogen regimes indicates potential agricultural applications for Cga1, including manipulation of biomass, chlorophyll/chloroplast content, and harvest index. These results indicate a conserved mechanism by which Cga1 regulates chloroplast development in higher plants.

A developmental transcriptional network for maize defines coexpression modules.

Here, we present a genome-wide overview of transcriptional circuits in the agriculturally significant crop species maize (Zea mays). We examined transcript abundance data at 50 developmental stages, from embryogenesis to senescence, for 34,876 gene models and classified genes into 24 robust coexpression modules. Modules were strongly associated with tissue types and related biological processes. Sixteen of the 24 modules (67%) have preferential transcript abundance within specific tissues. One-third of modules had an absence of gene expression in specific tissues. Genes within a number of modules also correlated with the developmental age of tissues. Coexpression of genes is likely due to transcriptional control. For a number of modules, key genes involved in transcriptional control have expression profiles that mimic the expression profiles of module genes, although the expression of transcriptional control genes is not unusually representative of module gene expression. Known regulatory motifs are enriched in several modules. Finally, of the 13 network modules with more than 200 genes, three contain genes that are notably clustered (P < 0.05) within the genome. This work, based on a carefully selected set of major tissues representing diverse stages of maize development, demonstrates the remarkable power of transcript-level coexpression networks to identify underlying biological processes and their molecular components.

Genome-wide expression profiling of maize in response to individual and combined water and nitrogen stresses.

BACKGROUND: Water and nitrogen are two of the most critical inputs required to achieve the high yield potential of modern corn varieties. Under most agricultural settings however they are often scarce and costly. Fortunately, tremendous progress has been made in the past decades in terms of modeling to assist growers in the decision making process and many tools are now available to achieve more sustainable practices both environmentally and economically. Nevertheless large gaps remain between our empirical knowledge of the physiological changes observed in the field in response to nitrogen and water stresses, and our limited understanding of the molecular processes leading to those changes. RESULTS: This work examines in particular the impact of simultaneous stresses on the transcriptome. In a greenhouse setting, corn plants were grown under tightly controlled nitrogen and water conditions, allowing sampling of various tissues and stress combinations. A microarray profiling experiment was performed using this material and showed that the concomitant presence of nitrogen and water limitation affects gene expression to an extent much larger than anticipated. A clustering analysis also revealed how the interaction between the two stresses shapes the patterns of gene expression over various levels of water stresses and recovery. CONCLUSIONS: Overall, this study suggests that the molecular signature of a specific combination of stresses on the transcriptome might be as unique as the impact of individual stresses, and hence underlines the difficulty to extrapolate conclusions obtained from the study of individual stress responses to more complex settings.

The rice R2R3-MYB transcription factor OsMYB55 is involved in the tolerance to high temperature and modulates amino acid metabolism.

Temperatures higher than the optimum negatively affects plant growth and development. Tolerance to high temperature is a complex process that involves several pathways. Understanding this process, especially in crops such as rice, is essential to prepare for predicted climate changes due to global warming. Here, we show that OsMYB55 is induced by high temperature and overexpression of OsMYB55 resulted in improved plant growth under high temperature and decreased the negative effect of high temperature on grain yield. Transcriptome analysis revealed an increase in expression of several genes involved in amino acids metabolism. We demonstrate that OsMYB55 binds to the promoter regions of target genes and directly activates expression of some of those genes including glutamine synthetase (OsGS1;2) glutamine amidotransferase (GAT1) and glutamate decarboxylase 3 (GAD3). OsMYB55 overexpression resulted in an increase in total amino acid content and of the individual amino acids produced by the activation of the above mentioned genes and known for their roles in stress tolerance, namely L-glutamic acid, GABA and arginine especially under high temperature condition. In conclusion, overexpression of OsMYB55 improves rice plant tolerance to high temperature, and this high tolerance is associated with enhanced amino acid metabolism through transcription activation.

GNC and CGA1 modulate chlorophyll biosynthesis and glutamate synthase (GLU1/Fd-GOGAT) expression in Arabidopsis.

Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light, nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination, elongation growth and flowering time. In this work, we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4, HEMA1, PORB, and PORC). As previously demonstrated, the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination, leaf production, flowering time and senescence. In contrast, the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence, results indicate that regulation of both GNC and CGA1 by light, nitrogen, cytokinin, and GA acts to modulate nitrogen assimilation, chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology.